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FilmArray, an automated nested multiplex PCR system for multi-pathogen detection: development and application to respiratory tract infection.

Poritz MA, Blaschke AJ, Byington CL, Meyers L, Nilsson K, Jones DE, Thatcher SA, Robbins T, Lingenfelter B, Amiott E, Herbener A, Daly J, Dobrowolski SF, Teng DH, Ririe KM - PLoS ONE (2011)

Bottom Line: FilmArray has the potential to detect greater than 100 different nucleic acid targets at one time.Validation of the FilmArray technology was achieved through development of a panel of assays capable of identifying 21 common viral and bacterial respiratory pathogens.We demonstrate that automated identification of pathogens from their corresponding target amplicon(s) can be accomplished by analysis of the DNA melting curve of the amplicon.

View Article: PubMed Central - PubMed

Affiliation: Idaho Technology, Inc., Salt Lake City, Utah, United States of America. mark_poritz@idahotech.com

ABSTRACT
The ideal clinical diagnostic system should deliver rapid, sensitive, specific and reproducible results while minimizing the requirements for specialized laboratory facilities and skilled technicians. We describe an integrated diagnostic platform, the "FilmArray", which fully automates the detection and identification of multiple organisms from a single sample in about one hour. An unprocessed biologic/clinical sample is subjected to nucleic acid purification, reverse transcription, a high-order nested multiplex polymerase chain reaction and amplicon melt curve analysis. Biochemical reactions are enclosed in a disposable pouch, minimizing the PCR contamination risk. FilmArray has the potential to detect greater than 100 different nucleic acid targets at one time. These features make the system well-suited for molecular detection of infectious agents. Validation of the FilmArray technology was achieved through development of a panel of assays capable of identifying 21 common viral and bacterial respiratory pathogens. Initial testing of the system using both cultured organisms and clinical nasal aspirates obtained from children demonstrated an analytical and clinical sensitivity and specificity comparable to existing diagnostic platforms. We demonstrate that automated identification of pathogens from their corresponding target amplicon(s) can be accomplished by analysis of the DNA melting curve of the amplicon.

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Amplification and melt curves at low target levels.Respiratory Pathogen pouches were injected with viral transport medium spiked with 1 TCID50 of the FluA- H1 seasonal virus used in Figure 4, and run on the FilmArray instrument. Real-time amplification curves (A) and post-amplification melt curves (B) for selected wells on the array are shown. Assays are spotted in triplicate: FluA-pan1 (orange), FluA-pan2 (pink), FluA-H1-pan (red). The ordinate scales are the same as in Figure 4.
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pone-0026047-g006: Amplification and melt curves at low target levels.Respiratory Pathogen pouches were injected with viral transport medium spiked with 1 TCID50 of the FluA- H1 seasonal virus used in Figure 4, and run on the FilmArray instrument. Real-time amplification curves (A) and post-amplification melt curves (B) for selected wells on the array are shown. Assays are spotted in triplicate: FluA-pan1 (orange), FluA-pan2 (pink), FluA-H1-pan (red). The ordinate scales are the same as in Figure 4.

Mentions: A diagnostic system that automates the technically demanding steps of nucleic acid isolation and PCR amplification would benefit from automated analysis of the PCR results. FilmArray runs generate large amounts of data in the form of real time amplification curves and the associated melt curves. In similar systems the properties of the amplification curve are used to make a positive or negative call for that assay [65]. In the course of analyzing the FilmArray data from reference strains and clinical samples, we observed that the amplicon melt curve shapes and Tms were highly specific to the organism targeted by the nested PCR and thus could provide an additional filter for detecting each organism. For high or moderate titer organisms, the FilmArray system produces both robust amplification and melt curves with a high signal-to-noise ratio (Figure 4A and B). For very low titer organisms, the amplification curve is often obscured by the noise inherent in thermocycling. For example, Figure 6 shows the amplification and melt curve data from a pouch injected with a very low level FluA sample (1/200th of that in Figure 4). Of the three assays in the pouch that can detect this organism, neither the FluA pan2 nor the FluA H1 pan amplification curves show a rise above baseline; only the FluA pan1 assay produced significant amplification. By contrast, a robust signal can be detected in the melt curves for the same sample (Figure 6B). The automated analysis of the melt curves produced a positive result for the FluA pan1 and pan2 assays (2 or more of the 3 replicates were positive) and a negative result for the FluA H1 pan assay (only one melt curve was positive).


FilmArray, an automated nested multiplex PCR system for multi-pathogen detection: development and application to respiratory tract infection.

Poritz MA, Blaschke AJ, Byington CL, Meyers L, Nilsson K, Jones DE, Thatcher SA, Robbins T, Lingenfelter B, Amiott E, Herbener A, Daly J, Dobrowolski SF, Teng DH, Ririe KM - PLoS ONE (2011)

Amplification and melt curves at low target levels.Respiratory Pathogen pouches were injected with viral transport medium spiked with 1 TCID50 of the FluA- H1 seasonal virus used in Figure 4, and run on the FilmArray instrument. Real-time amplification curves (A) and post-amplification melt curves (B) for selected wells on the array are shown. Assays are spotted in triplicate: FluA-pan1 (orange), FluA-pan2 (pink), FluA-H1-pan (red). The ordinate scales are the same as in Figure 4.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3198457&req=5

pone-0026047-g006: Amplification and melt curves at low target levels.Respiratory Pathogen pouches were injected with viral transport medium spiked with 1 TCID50 of the FluA- H1 seasonal virus used in Figure 4, and run on the FilmArray instrument. Real-time amplification curves (A) and post-amplification melt curves (B) for selected wells on the array are shown. Assays are spotted in triplicate: FluA-pan1 (orange), FluA-pan2 (pink), FluA-H1-pan (red). The ordinate scales are the same as in Figure 4.
Mentions: A diagnostic system that automates the technically demanding steps of nucleic acid isolation and PCR amplification would benefit from automated analysis of the PCR results. FilmArray runs generate large amounts of data in the form of real time amplification curves and the associated melt curves. In similar systems the properties of the amplification curve are used to make a positive or negative call for that assay [65]. In the course of analyzing the FilmArray data from reference strains and clinical samples, we observed that the amplicon melt curve shapes and Tms were highly specific to the organism targeted by the nested PCR and thus could provide an additional filter for detecting each organism. For high or moderate titer organisms, the FilmArray system produces both robust amplification and melt curves with a high signal-to-noise ratio (Figure 4A and B). For very low titer organisms, the amplification curve is often obscured by the noise inherent in thermocycling. For example, Figure 6 shows the amplification and melt curve data from a pouch injected with a very low level FluA sample (1/200th of that in Figure 4). Of the three assays in the pouch that can detect this organism, neither the FluA pan2 nor the FluA H1 pan amplification curves show a rise above baseline; only the FluA pan1 assay produced significant amplification. By contrast, a robust signal can be detected in the melt curves for the same sample (Figure 6B). The automated analysis of the melt curves produced a positive result for the FluA pan1 and pan2 assays (2 or more of the 3 replicates were positive) and a negative result for the FluA H1 pan assay (only one melt curve was positive).

Bottom Line: FilmArray has the potential to detect greater than 100 different nucleic acid targets at one time.Validation of the FilmArray technology was achieved through development of a panel of assays capable of identifying 21 common viral and bacterial respiratory pathogens.We demonstrate that automated identification of pathogens from their corresponding target amplicon(s) can be accomplished by analysis of the DNA melting curve of the amplicon.

View Article: PubMed Central - PubMed

Affiliation: Idaho Technology, Inc., Salt Lake City, Utah, United States of America. mark_poritz@idahotech.com

ABSTRACT
The ideal clinical diagnostic system should deliver rapid, sensitive, specific and reproducible results while minimizing the requirements for specialized laboratory facilities and skilled technicians. We describe an integrated diagnostic platform, the "FilmArray", which fully automates the detection and identification of multiple organisms from a single sample in about one hour. An unprocessed biologic/clinical sample is subjected to nucleic acid purification, reverse transcription, a high-order nested multiplex polymerase chain reaction and amplicon melt curve analysis. Biochemical reactions are enclosed in a disposable pouch, minimizing the PCR contamination risk. FilmArray has the potential to detect greater than 100 different nucleic acid targets at one time. These features make the system well-suited for molecular detection of infectious agents. Validation of the FilmArray technology was achieved through development of a panel of assays capable of identifying 21 common viral and bacterial respiratory pathogens. Initial testing of the system using both cultured organisms and clinical nasal aspirates obtained from children demonstrated an analytical and clinical sensitivity and specificity comparable to existing diagnostic platforms. We demonstrate that automated identification of pathogens from their corresponding target amplicon(s) can be accomplished by analysis of the DNA melting curve of the amplicon.

Show MeSH
Related in: MedlinePlus